Project Summary Eukaryotic cells employ several major DNA repair pathways to monitor and maintain the integrity of their genomes. Most past research has focused on characterization of the fundamental mechanisms and key proteins involved in these pathways after exposure of cells to exogenous chemicals or radiation. By contrast, the roles played by these repair systems in responding to the tens of thousands of DNA lesions that form spontaneously within cells each day is largely unexplored. In this proposed study, which we believe is the first of its kind, we will investigate the roles of all major pathways in maintaining cellular homeostasis during normal growth, i.e., in response to endogenous rather than exogenous sources of DNA damage. Our preliminary studies revealed that Saccharomyces cerevisiae (budding yeast) mutants defective in repair of DNA double-strand breaks (DSBs) by the homologous recombination (HR) pathway exhibit many phenotypes caused by unrepaired lesions. These phenotypes include a 3-fold increase in time spent in G2 phase, a characteristic dependent on the presence of functional DNA damage checkpoint genes, as well as strongly increased G2 DNA content revealed by FACS analysis, greatly enlarged cells, and striking changes in other morphological and physical properties. We subsequently screened mutants from a yeast deletion strain library to ask what other major repair pathways are most critical during normal growth. Mutants of only one other pathway, base excision repair (BER), also exhibited strong cellular stress responses. We propose to perform a series of experiments that will expand upon these findings by (1) characterizing the persistently activated DNA damage response in HR mutants using genetic and microscopy-based techniques, (2) performing critical tests of a model for persistent checkpoint activation in HR mutants, (3) characterizing the constitutive DNA damage signaling response in BER pathway mutants, and (4) performing experiments that will critically test the roles of reactive oxygen species (ROS) and stalled replication forks in generation of endogenous checkpoint- activating DNA lesions. Our major hypothesis is that defects in HR and BER, but not other pathways, lead to high levels of unrepaired lesions that stimulate a recurring checkpoint signaling cascade and many quantifiable physiological changes in cells. Since the major DNA repair pathways analyzed in this project are conserved in all eukaryotes including humans, the findings of the work will have strong relevance to the related fields of human carcinogenesis and cellular aging.